Receptors for extracellular nucleotides are the focus of increasing attention for their ability to cause release of plasma membrane vesicles (microparticles, MPs). Here, we show that monocyte-derived human dendritic cells (DCs) stimulated with a P2X 7 receptor (P2X 7 R) agonist undergo a large release of MPs endowed with procoagulant activity. Functional and Western blot studies revealed that MPs contain the membrane-bound form of tissue factor (TF), a glycoprotein acting as essential cofactor of activated factor VII and triggering blood coagulation. Quiescent DCs express the membrane-bound (full length), as well as truncated alternatively spliced TF forms. DC reactivity to anti-TF Abs disappeared almost completely on stimulation with ATP or benzoyl ATP (BzATP), as shown by immunoblot and confocal microscopy analysis. Concurrently, TF reactivity and activity appeared in the vesicular fraction, indicating that MPs are important carriers for the dissemination of full-length TF form. Activity of MP-bound TF, comparable to that of relipidated recombinant TF, was dose dependently inhibited by the addition of a specific anti-human TF antibody. We infer that a large fraction of this protein, and its procoagulant potential, are "deliverable" after physiological or pathological stimuli. These findings might have implications for triggering and propagating coagulation in healthy and atherosclerotic vessels.-. Stimulation of P2 (P2X 7 ) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles. FASEB J. 21, 1926 -1933 (2007)

Objective-We investigated in patients with ongoing myocardial infarction (MI) whether coagulation factor VII (FVII) and tissue factor (TF) levels are affected at admission by genetic components and whether they may predict subsequent cardiovascular events. Methods and Results-256 patients admitted for MI were evaluated for FVII and TF antigen levels before any treatment at entry, and were genotyped for FVII and TF polymorphisms. FVII gene insertions at Ϫ323, 11293 and the Ϫ402G/A change predicted FVII levels and explained 14% of variance. The Ϫ603 TF gene polymorphism failed to affect significantly TF levels (Pϭ0.07). These variables were correlated with the incidence of death (36 patients) and reinfarction (9 patients) after a median follow-up of 397 days. Events were independently predicted by FVII (HR 2.1, 95% CI 1.2 to 5.7) and TF (HR 4.1, 95% CI 2 to 11) levels. Composite end point was significantly worse when both parameters were above the receiver-operating characteristics (ROC) values (HR 8.3, 95% CI 5 to 18, compared with FVII and TF below), and above the ROC value of TF (Ͼ630 pg/mL) it differed among FVII genotype groups. Conclusions-Admission

Decreased ADAMTS13, particularly in MS patients with cerebral microbleeds, which deserves further investigation, and increased PAI-1 and TFPI levels were observed in MS patients, which deserves further investigation. No relationship between hemostasis plasma levels and measures of disease severity was detected.

Olivieri O. Activated factor VII-antithrombin complex predicts mortality in patients with stable coronary artery disease: a cohort study.J Thromb Haemost 2016; 14: 655-66. Essentials• Activated factor VII-antithrombin complex (FVIIa-AT) in plasma may reflect tissue factor exposure.• FVIIa-AT levels were assessed in an angiographically controlled coronary artery disease (CAD) cohort.• High FVIIa-AT levels correlated with an increased thrombin generation.• High FVIIa-AT levels were associated with a greater risk of mortality in patients with stable CAD.Summary. Background: Plasma concentration of activated factor VII (FVIIa)-antithrombin (AT) complex has been proposed as an indicator of intravascular exposure of tissue factor. Objectives: The aims of this observational study were to evaluate (i) FVIIa-AT plasma concentration in subjects with or without coronary artery disease (CAD) and (ii) its association with mortality in a prospective cohort of patients with CAD. Methods: FVIIa-AT levels were measured by ELISA in 686 subjects with (n = 546) or without (n = 140) angiographically proven CAD. Subjects with acute coronary syndromes and those taking anticoagulant drugs at the time of enrollment were excluded. CAD patients were followed for total and cardiovascular mortality. Results: There was no difference in FVIIa-AT levels between CAD (84.8 with 95% confidence interval [CI] 80.6-88.2 pmol L -1 ) and CAD-free subjects (83.9 with 95% CI 76.7-92.8 pmol L -1 ). Within the CAD population, during a 64-month median followup, patients with FVIIa-AT levels higher than the median value at baseline (≥ 79 pmol L -1 ) had a two-fold greater risk of both total and cardiovascular mortality. Results were confirmed after adjustment for sex, age, the other predictors of mortality (hazard ratio for total mortality: 2.05 with 95% CI 1.22-3.45, hazard ratio for cardiovascular mortality 1.94 with 95% CI 1.01-3.73, with a slight improvement of C-statistic over traditional risk factors), FVIIa levels, drug therapy at discharge, and even patients using all the usual medications for CAD treatment. High FVIIa-AT levels also correlated with increased thrombin generation. Conclusions: This preliminary study suggests that plasma concentration of FVIIa-AT is a thrombophilic marker of total and cardiovascular mortality risk in patients with clinically stable CAD.

SummaryWe characterized a symptomatic CRMred factor X (FX) deficiency produced by the Glu19Ala mutation in the γ-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19AlaFX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation.The study of this FX deficiency provides an “in vivo” and “in vitro” model for the investigation of Gla domain interactions.

BackgroundFew studies have so far investigated the relationship between apolipoprotein CIII (Apo CIII) and coagulation pathway in subjects with or without coronary artery disease (CAD).Methods and ResultsSerum Apo CIII concentrations and plasma coagulant activities of factor II (FII:c), factor V (FV:c), and factor VIII (FVIII:c), and activated factor VII (FVIIa) were analyzed in a total of 933 subjects, with (n=687) or without (n=246) angiographically demonstrated CAD and not taking anticoagulant drugs. Activated factor X (FXa) generation assay was performed on plasma from subgroups of subjects with low and high levels of Apo CIII. A statistical incremental concentration of FII:c, FV:c, and FVIIa levels was observed through the quartiles of Apo CIII distribution in the population considered as a whole. Significant results were confirmed for FII:c in CAD and CAD‐free subgroup when separately considered. Subjects within the highest Apo CIII quartile (>12.6 mg/dL) had high FII:c levels not statistically different from those of carriers of 20210A allele (n=40; 4.28%). In a multiple linear model, Apo CIII was the best predictor of FII:c variability, after adjustment for age, gender, plasma lipids, CRP, creatinine, diagnosis, and carriership of 20210A allele. FXa generation was increased and its lag time shortened in plasmas with high Apo CIII levels. However, after thrombin inhibition by hirudin, differences between low and high Apo C‐III samples disappeared.ConclusionsElevated concentrations of Apo CIII are associated with an increase of thrombin activity to an extent comparable with the carriership of G20210A gene variant and mainly modulating the thrombin generation.

The carboxyl-terminal region is NOT essential for secreted and functional levels of coagulation factor X. J Thromb Haemost 2015; 13: 1468-74.Summary. Background: The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl-terminus, with that of FX being the most extended. This region is essential for FVII, FIX and PC secretion. Objectives: To provide experimental evidence for the role of the FX carboxyl-terminus. Methods: Recombinant FX (rFX) variants were expressed in multiple eukaryotic cell systems. Protein and activity levels were evaluated by ELISA, coagulant and amidolytic assays. Results and discussion: Expression of a panel of progressively truncated rFX variants in HEK293 cells revealed that the deletion of up to 21 residues in the carboxyl-terminus did not significantly affect secreted protein levels, as confirmed in HepG2 and BHK21 cells. In contrast, chimeric rFX-FVII variants with swapped terminal residues showed severely reduced levels. The truncated rFX variants revealed normal amidolytic activity, suggesting an intact active site. Intriguingly, these variants, which included that resembling the activated FXb form once cleaved, also displayed remarkable or normal pro-coagulant capacity in PT-and aPTT-based assays. This supports the hypothesis that subjects with nonsense mutations in the FX carboxyl-terminus, so far never identified, would be asymptomatic. Conclusions: For the first time we demonstrate that the FX carboxyl-terminal region downstream of residue K467 is not essential for secretion and provides a modest contribution to procoagulant properties. These findings, which might suggest an involvement of the carboxyl-terminal region in the divergence of the homologous FX, FVII, FIX and PC, help to interpret the mutational pattern of FX deficiency.

BackgroundFactor XII (FXII) activation initiates the intrinsic (contact) coagulation pathway. It has been recently suggested that FXII could act as an autoimmunity mediator in multiple sclerosis (MS). FXII depositions nearby dentritic cells were detected in the central nervous system of MS patients and increased FXII activity has been reported in plasma of relapsing remitting and secondary progressive MS patients. FXII inhibition has been proposed to treat MS.ObjectiveTo investigate in MS patients multiple FXII-related variables, including the circulating amount of protein, its pro-coagulant function, and their variation over time. To explore kinetic activation features of FXII in thrombin generation (TG).MethodsIn plasma from 74 MS patients and 49 healthy subjects (HS), FXII procoagulant activity (FXII:c) and FXII protein (FXII:Ag) levels were assessed. Their ratio (FXII:ratio) values were derived. Intrinsic TG was evaluated by different triggers.ResultsHigher FXII:Ag levels (p = 0.003) and lower FXII:ratio (p < 0.001) were detected in MS patients compared with HS. FXII variables were highly correlated over four time points, which supports investigation of FXII contribution to disease phenotype and progression. A significant difference over time was detected for FXII:c (p = 0.031). In patients selected for the lowest FXII:ratio, TG triggered by ellagic acid showed a trend in lower endogenous thrombin potential (ETP) in MS patients compared with HS (p = 0.042). Intrinsic triggering of TG by nucleic acid addition produced longer time parameters in patients than in HS and substantially increased ETP in MS patients (p = 0.004) and TG peak height in HS (p = 0.008). Coherently, lower FXII:ratio and longer lag time (p = 0.02) and time to peak (p = 0.007) point out a reduced response of FXII to activation in part of MS patients.ConclusionIn MS patients, factor-specific and modified global assays suggest the presence of increased FXII protein level and reduced function within the intrinsic coagulation pathway. These novel findings support further investigation by multiple approaches of FXII contribution to disease phenotype and progression.